Diagnosis and treatment of AIDS onset

ABSTRACT

The invention provides assays for the presence or absence of CD4+ T cell subpopulations carrying particular Vβ components of the T cell receptor (TCR-Vβ) in persons infected with HIV, including amplification of mRNA from T cells with primers specific to each TCR-Vβ to detect the presence or absence of each TCR-Vβ in a sample and primers for use in such amplification assays are disclosed. The invention also provides assays of antibody-containing fluids of a person infected with HIV to determine the immunodeficiency where the fluid is suspected to contain an antibody having a paratope specific to an epitope on a TCR-Vβ. The invention also provides a binding agent specific to a paratope where the paratope is specific to an epitope on a TCR-Vβ. The invention also provides a method of assay of the fluids of a person infected with HIV to determine the immunodeficiency of the person which utilizes a binding agent specific to complexes containing anti-TCR-Vβ antibody bound to TCR-Vβ. The invention also provides a method of treatment of a person infected with HIV to attenuate or avert immunodeficiency which utilize a binding agent that is homologous with an epitope on TCR-Vβ. The invention also provides a method of treatment of a person infected with HIV to attenuate or avert immunodeficiency which involves removing an antibody capable of binding to an epitope on TCR-Vβ from the blood of a person infected with HIV. Finally, the invention provides a method of vaccination of a person infected or at risk for infection with HIV which raises antiidiotypic antibodies specific to free antibodies containing a paratope specific to an epitope on a TCR-Vβ.

This is a continuation of application Ser. No. 07/973,485, filed Nov. 9,1992, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention involves a method of assay of CD4+ T cells of aperson infected with HIV to determine the immunodeficiency of theperson. More specifically, the invention relates to a method of assayfor the extent of depletion of subpopulations of CD4+ T cells whichexpress specific β chains of the T cell receptor. The invention alsoinvolves additional assays, products, and methods of vaccination relatedto the above.

2. Description of Background Art

The importance of T cell counts in HIV infected individuals has alreadybeen recognized. Currently, the National Institute of Allergies andInfectious Diseases (NIAID) has recommended that the CD4+ T cell countsin HIV-infected patients be determined two to four times per year.Healthy individuals normally have 800-1000 CD4+ cells/μl of blood. NIAIDrecommends that AZT treatment begin when CD4 levels drops to 500 CD4+cells/μl and that when levels drop below 200 CD4+ cells/μl that AZT bediscontinued and pentamidine therapy be undertaken. CD4+ cell counts arecurrently performed using flow cytometry. FDA approval has recently beensought for a method of counting CD4+ cells by solubilizing CD4 receptorsand quantifying by means of a microtiter colorimetric immunoassay.

The cause of T cell depletion in HIV infected individuals has remained amystery, however. It is unlikely that T cell depletion is a directresult of HIV infection. Although HIV is known to infect CD4+ T cells bybinding to the surface protein, only a fraction of CD4+ T cells, around1 in 10000 are actually infected with the virus. A number of theorieshave been proposed to explain the depletion phenomenon but none havebeen proven or disproved.

One theory is that HIV requires the presence of another pathogen todeplete immune cell populations. The second pathogen may beopportunistic and depress the immune system simply by its presence. Thesecond pathogen may also work as a "co-factor" working in concert withthe HIV virus to directly suppress T cell populations. Mycoplasmas havebeen proposed as one candidate for co-factor pathogen status.

A second theory is that HIV triggers an autoimmune reaction thatdestroys immune cells. There is no agreement as to which of the possiblemarkers on immune cells provides the binding site for the autoimmuneantibodies. One group has proposed that antibodies to HIV envelopeprotein (gp120) could recognize and bind to the major histocompatibility(MHC) proteins. These anti-MHC antibodies would then cause the depletionof the T cells.

A third theory is that HIV-infected T cells fuse with uninfected cellsto produce syncytia consisting of non-functioning cells. Thus, a singleHIV-infected cell could bring about the demise of many non-infected Tcells. The observation of syncytia formation was made in vitro, and noin vivo results have yet been published to substantiate this hypothesis.

Finally, some researchers have proposed that T cell depletion is causedby a HIV protein acting as a superantigen. Normally, when antigen ispresented to a T cell, interaction of the alpha and beta chains of the Tcell receptor with antigen is required for T cell activation.Superantigens interact only with the beta chain and are able to bypassthe requirement of alpha chain interaction. By removing the alpha chainrequirement, superantigens are able to activate a much larger proportionof the T cell subpopulation. Furthermore, T cells stimulated bysuperantigens eventually lose their ability to respond to antigens andundergo a programmed cell death known as apoptosis.

There is no agreement as to which HIV protein might have superantigenproperties and, indeed, there has been no direct proof of superantigeninvolvement in T cell depletion. Some researchers have proposed that HIVgp120 may be the superantigen. Others have proposed that the nef geneproduced by an open reading frame at the 3' end of the IHV genome may bethe superantigen.

It is an object of the invention to delineate the pattern of T celldepletion in HIV infected individuals in order to develop effectiveintervention therapies to slow or halt T cell depletion and to moreeffectively time preventive treatment for opportunistic diseases.

SUMMARY OF THE INVENTION

The invention relates to a method of assay of CD4+ T cells of a personinfected with HIV, preferably HIV-1, to determine the immunodeficiencyof the person. This method involves measuring the extent of depletion ofa CD4+ Tcell subtype. The subtype is defined by the expression of aparticular beta chain of the T cell receptor, having a constant regionand a variable region (the variable region hereinafter designated TCR-Vβor Vβ). The selection of the TCR-Vβs may be any of the TCR-Vβs,including TCR-Vβ14, TCR-Vβ15, TCR-Vβ16, TCR-Vβ17, TCR-β18, TCR-Vβ19, orTCR-Vβ20.

In one embodiment of the foregoing assay a sample from the HIV infectedperson is isolated. The sample is prepared for contact with a bindingagent specific to an analog of TCR-β. The analog may be TCR-Vβ, afragment of TCR-Vβ, mRNA encoding TCR-Vβ, mRNA encoding a fragment ofTCR-Vβ, genomic DNA encoding TCR-Vβ, genomic DNA encoding a fragment ofTCR-Vβ, cDNA encoding TCR-β, or cDNA encoding a fragment of TCR-Vβ. Theprepared sample is contacted with the binding agent under conditionssuitable for binding of the binding agent and the analog. The extent ofthe binding is determined and a correlation is made to the extent of thedepletion.

The binding agent in the foregoing assay may be an antibody, a fragmentof an antibody, a binding receptor or a binding receptor fragment. Thebinding agent may also be a nudeic acid probe where the analog is in theform of mRNA, genomic DNA or cDNA.

Another embodiment of the foregoing assay involves isolating a samplefrom the HIV infected person. The sample is then prepared for contactwith a first primer and a second primer. The first primer issubstantially homologous to genomic DNA, MRNA or cDNA encoding a portionof the constant region of the TCR-β chain and the second primer issubstantially homologous to genomic DNA, mRNA or cDNA encoding a portionof the variable region of the TCR-β chain. The prepared sample iscontacted with the first and second primers and amplification enzymesunder conditions suitable for amplification of the analog. The extent ofthe amplification is determined and a correlation is made to the extentof the depletion.

The first primer may be GTGCACCTCCTTCCCATT (SEQ ID NO:1) and the secondprimer may be any one of the following:

(a) GCACAACAGTTCCCTGACTTGCAC(SEQ ID NO:2);

(b) TCATCAACCATGCAAGCCTGACCT(SEQ ID NO:3);

(c) GTCTCTAGAGAGAAGAAGGAGCGC(SEQ ID NO:41);

(d) ACATATGAGAGTGGATTTGTCATT(SEQ ID NO:5);

(e) ATACTTCAGTGAGACACAGAGAAAC(SEQ ID NO:6);

(f) TTCCCTAACTATAGCTCTGAGCTG(SEQ ID NO:7);

(g) AGGCCTGAGGGATCCGTCTC(SEQ ID NO:8);

(h) CCTGAATGCCCCAACAGCTCTC(SEQ ID NO:9);

(i) ATTTACTTTAACAACAACGTTCCG(SEQ ID NO:10);

(j) CCTAAATCTCCAGACAAAGCTCAC(SEQ ID NO:11);

(k) CTCCAAAAACTCATCCTGTACCTT(SEQ ID NO:12);

(l) TCAACAGTCTCCAGAATAAGGACG(SEQ ID NO:12);

(m) AAAGGAGAAGTCTCAGAT(SEQ ID NO:14);

(n) CAAGGAGAAGTCCCCAAT(SEQ ID NO:15);

(o) GGTGAGGGTACAACTGCC(SEQ ID NO:16);

(p) GTCTCTCGAAAAGAGAAGAGGAAT(SEQ ID NO:17);

(q) AGTGTCTCTCGACAGGCACAGGCT(SEQ ID NO:18);

(r) AAAGAGTCTAAACAGGATGAGTCC(SEQ ID NO:19);

(S) CAGATAGTAAATGACTTTCAG(SEQ ID NO:20);

(t) GATGAGTCAGGAATGCCAAAGGAA(SEQ ID NO:21);

(u) CAATGCCCCAAGAACTCACCCTGC(SEQ ID NO:22);

(V) AGCTCTGAGGTGCCCCAGAATCTC(SEQ ID NO:23);

(w) TTCTGCAGAGAGGCTCAAAGGACT(SEQ ID NO:24);

(x) CTCAGTTGAAAGGCCTGATGGATC(SEQ ID NO:25);

(y) CTCAGCTCAACAGTTCAGTGACTA(SEQ ID NO:26); and

(z) CCAATCCAGGAGGCCGAACACTTC(SEQ ID NO:27).

The invention further relates to a method of assay of anantibody-containing fluid of a person infected with HIV to determine theimmunodeficiency of the person. The method involves isolating a samplefrom the HIV-infected person, where the sample is suspected to containan antibody having a paratope specific to the variable region of a TCR-βchain (TCR-Vβ). The sample is prepared for contact with a binding agentspecific to the paratope of the antibody. The prepared sample iscontacted with the binding agent under conditions suitable for bindingof the antibody with the binding agent. The extent of the binding isdetermined and a correlation is made to the extent of the depletion. Ina preferred embodiment of the foregoing assay, the paratope of theantibody may be specific to an epitope on any one of the following:Ser-Ala-Val-Ile-Ser-Gln-Lys-Pro-Ser-Arg-Asp-Ile-Cys-Gln-Arg-Gly-Thr-Ser-Leu-Thr(SEQID NO:28);Gln-Leu-Gln-Glu-Thr-Glu-Asn-His-Lys-Lys-Arg-Phe-Ser-Ser-Gln-Cys-Pro(SEQID NO:29);Gln-Asn-Leu-Ser-Ala-Ser-Arg-Pro-Gln-Asp-Arg-Gln-Phe-Ile-Leu-Ser-Ser-Lys(SEQID NO:30);Asp-Gly-Tyr-Ser-Val-Ser-Arg-Ser-Lys-Thr-Glu-Asp-Phe-Leu-Leu(SEQ IDNO:31);Pro-Arg-Asn-Arg-Ile-Thr-Lys-Ile-Gly-Lys-Arg-Ile-Met-Leu-Glu-Cys(SEQ IDNO:32); orPro-Arg-His-Leu-Val-Arg-Arg-Arg-Gly-Gln-Glu-Ala-Arg-Leu-Arg-Cys(SEQ IDNO:33). The paratope may alternatively be specific to an epitope on aTCR-Vβ which is one of the following: TCR-Vβ14, TCR-Vβ15, TCR-Vβ16,TCR-Vβ17, TCR-Vβ18, TCR-Vβ19, and TCR-Vβ20. The paratope of theantibody, in addition to the above specificities, may also be specificto an epitope on gp120 of HIV. In another embodiment of the foregoingassay, the binding agent may be an antibody, an antibody fragment, abinding receptor, or a polypeptide containing fewer than 100 aminoacids.

The invention further relates to a binding agent specific to a paratopeon a human antibody. The paratope on the antibody is specific to anepitope on a TCR-Vβ. The binding agent may be an antibody or an antibodyfragment. The antibody may be a binding receptor or a binding receptorfragment. The binding agent may also be described as a polypeptidecontaining fewer than 100 amino acids. In a preferred embodiment, theepitope on the binding agent may be on an amino acid sequence which isone of the following:Ser-Ala-Val-Ile-Ser-Gln-Lys-Pro-Ser-Arg-Asp-Ile-Cys-Gln-Arg-Gly-Thr-Ser-Leu-Thr(SEQID NO:28);Gln-Leu-Gln-Glu-Thr-Glu-Asn-His-Lys-Lys-Arg-Phe-Ser-Ser-Gln-Cys-Pro;Gln-Asn-Leu-Ser-Ala-Ser-Arg-Pro-Gln-Asp-Arg-Gln-Phe-Ile-Leu-Ser-Ser-Lys;Asp-Gly-Tyr-Ser-Val-Ser-Arg-Ser-Lys-Thr-Glu-Asp-Phe-Leu-Leu;Pro-Arg-Asn-Arg-Ile-Thr-Lys-Ile-Gly-Lys-Arg-Ile-Met-Leu-Glu-Cys; orPro-Arg-His-Leu-Val-Arg-Arg-Arg-Gly-Gln-Glu-Ala-Arg-Leu-Arg-Cys.

The invention further relates to a method of assay of the fluids of aperson infected with HIV to determine the immunodeficiency of theperson. The method involves isolating a sample from the HIV infectedperson, where the sample is suspected to contain an antibody having aparatope specific to an epitope on a TCR-Vβ and being bound to theTCR-Vβ by the paratope to form an antibody-TCR-Vβ pair. The sample isprepared for contact with a binding agent which selectively binds to theantibody-TCR-Vβ pair. The binding agent does not bind to the antibodywhen the antibody is not bound to TCR-Vβ and the binding agent does notbind to the TCR-Vβ when the antibody is not bound to TCR-Vβ. Theprepared sample is contacted with the binding agent under conditionssuitable for binding of the binding agent to any TCR-Vβ-pair. The extentof the binding is determined and a correlation is made to the presenceor amount of such antibody-TCRV-β pairing.

The invention further relates to a method of treatment of a personinfected with HIV to attenuate or avert immunodeficiency in the personinfected with HIV. The method involves inoculating the person infectedwith HIV with a binding agent which is substantially homologous with anepitope on a TCR-Vβ and which is reactive with a paratope on a freeantibody in the person's blood, where the paratope is specific to theepitope on the TCR-Vβ. The binding agent may be a polypeptide, a bindingreceptor or a binding receptor fragment. The binding agent may also bean antiidiotypic antibody or an antiidiotypic antibody fragment. Anantiidiotypic antibody is specific to the paratope of another antibody(ie. binding of the antiidiotypic antibody prevents the other antibodyfrom being able to bind to an epitope). Antiidiotypic antibody fragmentsuseful in the invention are those fragments of antiidiotypic antibodieswhich possess the ability to prevent an antibody from being able to bindto an epitope.

The invention further relates to another method of treatment of a personinfected with HIV to attenuate or avert immunodeficiency. The methodinvolves removing blood from the person infected with HIV; removingantibody from the blood of the person, the antibody having a paratopespecific to an epitope present on TCR-Vβ and reintroducing the bloodinto the person.

The invention finally relates to a method of vaccination of a personinfected with HIV or a person at risk for infection with HIV in order toattenuate or avert immunodeficiency. The method involves inoculatingperson with an immunogenic substance capable of raising antiidiotypicantibodies in the person. The antiidiotypic antibodies are specific tofree antibodies containing a paratope specific to an epitope on aTCR-Vβ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of the T cell receptor Vβ genes in T cellsfrom HIV infected individuals and from healthy controls.

FIG. 2 shows the expression of the T cell receptor Vα genes in T cellsfrom HIV infected individuals and from healthy controls.

FIG. 3 shows the results of the DEIA assay of the amplified Vβtranscripts obtained with T cells from a control and the results ofSouthern blot analysis of amplified Cβ and Vβ transcripts obtained withT cells from HIV infected individuals.

FIG. 4 shows the expression of T cell receptor Vβ genes in T cells fromtwo groups of HIV infected patients at different stages of infection(CDC Stages II and III).

FIG. 5 shows the results of cytofluorimetric analysis of cell surfaceCD3 and CD4 modulation in four healthy donors, after the stimulation oflymphocytes with a mixture of Staphylococcal enterotoxins B, C2 and E,and an overnight incubation with HIV gp120.

FIG. 6 shows the results of cytofluorimetric analysis of cell surfaceVβ6 and Vβ8 modulation in four healthy donors, after the stimulation oflymphocytes with a mixture of Staphylococcal enterotoxins B, C2 and E,and an overnight incubation with HIV gp120.

FIG. 7 shows the results of cytofluorimetric analysis of cell surfaceVβ5, Vβ6, Vβ8 and Vβ12 expression in four healthy donors, after anovernight incubation of lymphocytes with two doses of HIV gp120 and asubsequent stimulation of the cells with a mixture of Staphylococcalenterotoxins B, C2 and E.

FIG. 8 shows the calculated hydropathic profiles of deduced amino acidsequences of the Vβ4, Vβ19 and Vβ20 peptides.

FIG. 9 shows the immune reactivity of sera obtained from normal donorsand from HIV-infected patients at CDC stage IV against theimmunodominant fragments of Vβ4, Vβ19 and Vβ20 peptides.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method of assay of CD4+ T cells of a personinfected with a Human Immunodeficiency Virus (HIV) to determine theimmunodeficiency of the person. In a preferred embodiment, HIV is HIV-1.HIV-1 is also known as HTLV-III or LAV. This method involves measuringthe extent of depletion of a CD4+ Tcell subtype as defined by theexpression of a particular TCR-Vβ on the surface of the T cell as acomponent of the T cell receptor. The selection of the TCR-Vβs mayinclude any TCR-Vβ subtype. A TCR-Vβ subtype is defined as any singlemember of a TCR-Vβ subfamily (eg. TCR-Vβ1, TCR-Vβ5.1, TCR-Vβ5.2, etc.).It is preferred to include the TCR-Vβ14, TCR-Vβ15, TCR-Vβ16, TCR-Vβ17,TCR-Vβ18, TCR-Vβ19, or TCR-Vβ20 subtypes in the assay.

In one embodiment of the foregoing assay a sample from the HIV infectedperson is isolated. The sample is prepared for contact with a bindingagent specific to a TCR-Vβ subtype which may be the TCR-Vβ protein, afragment of the TCR-Vβ protein, mRNA encoding the TCR-Vβ, mRNA encodinga fragment of the TCR-Vβ, genomic DNA encoding TCR-Vβ, genomic DNAencoding a fragment of TCR-Vβ, cDNA encoding TCR-Vβ, or cDNA encoding afragment of TCR-Vβ. The sample is preferably blood, although any fluidcontaining a representative population of CD4+ T cells will work withthe method. The prepared sample is contacted with the binding agentunder conditions suitable for binding of the binding agent and theanalog. The extent of the binding is determined and a correlation ismade to the extent of the depletion.

Where the analog is the TCR-Vβ protein or a fragment of the TCR-Vβprotein the binding agent in the foregoing assay may be an antibody, afragment of an antibody, a binding receptor, or a binding receptorfragment which selectively recognize and bind to portions of the TCR-Vβprotein. If a fragment of an antibody is used as the binding agent, theonly requirement of the fragment is that the fragment is able tospecifically recognize and bind to any one TCR-Vβ subtype. A receptormay be any non-immunoglobulin protein which is ordinarily found on thesurface of any cell type. The receptor may be prepared by anyconventional means which involves freeing the receptor from the cellmembrane and isolating the receptor in a substantially pure form.Alternatively, the receptor may be prepared by recombinant DNA methods,including producing the receptor in a transformed host and isolating thereceptor in a substantially pure form. The analog may be the TCR-Vβprotein which is present on intact T cells. In this case, flow cytometrymay be used to determine the extent to the binding of the analog. Theanalog may also be the TCR-Vβ protein or a fragment of the TCR-Vβprotein which has been released from T cells by solubilization of themembranes, by peptidase treatment of the cells or other methods known inthe art. The extent of binding in this case may be determined byimmunoassays known in the art, including sandwich immunoassays,competitive immunoassays or other assay formats known in the art.

The binding agent may also be a nucleic acid probe where the analog isin the form of mRNA, genomic DNA or cDNA. The sequence of the probe issubstantially homologous to the sequence of any one TCR-Vβ subtype andwill preferentially hybridize to any nucleic acid encoding the same typeof specific TCR-Vβ under appropriate hybridization conditions. Methodsof predicting appropriate hybridization conditions or determining suchconditions are known in the art. The probe may be labelled by anyconventional method, including labelling with ³² P- or ³⁵ S- taggednucleotides at one end or throughout the probe, labelling withbiotin-tagged nucleotides, and chemiluminescent labelling.Alternatively, a second probe may be utilized in a sandwich or otherformat to detect the binding of the above probe.

Where the analog is mRNA or cDNA which is prepared from the total RNA ormRNA present in a sample, there is no need to isolate subpopulations ofcells from the sample. However, it is preferable to isolate peripheralblood mononuclear cells and isolate total RNA from these cells prior topreparing cDNA. Where the analog is genomic DNA, any cell which does notexpress a specific TCR-Vβ may be removed from the sample so that thegenomic DNA is indicative of the presence of the specific TCR-Vβsubtype.

Another embodiment of the foregoing assay involves isolating a samplefrom the HIV infected person and preparing the sample for contact with afirst primer and a second primer. A preferred method of preparation isperformed by isolation of peripheral blood mononuclear cells, isolationof total RNA from the cells and first strand synthesis of cDNA. Theprimers are oligonucleotides which can be prepared on commerciallyavailable synthesis machines or ordered from a company providing suchservices. The oligonucleotides are preferably DNA, although any nucleicacid may be used provided that the primer is able to hybridize to thenucleic acid analog and is also able to function in amplificationreactions.

The first primer is substantially homologous to genomic DNA, mRNA orcDNA encoding a portion of the constant region of the TCR-β chain andthe second primer is substantially homologous to genomic DNA, mRNA orcDNA encoding a portion of the variable region of the TCR-β chain. Thesecond primer must be capable of selectively binding to a sequencewithin any TCR-Vβ coding sequence in order to insure that only nudeicacids encoding TCR-Vβ of the desired subtype are amplified in thefollowing steps. The prepared sample is contacted with the first andsecond primers and amplification enzymes under conditions suitable foramplification of the analog. In a preferred embodiment, amplification isperformed using the polymerase chain reaction (PCR) utilizing Vβ- andCβ-specific primers shown in Table II. Other amplification techniqueswhich use the analog as a template and are capable of reproducing aportion of the analog or its corresponding opposite strand, such as theligase chain reaction, may also be employed. The extent of theamplification is determined and a correlation is made to the extent ofthe depletion. The extent of amplification may also be determined usinga DNA Enzyme Immunoassay (DEIA) kit available from Sorin BiomedicaS.p.A. (Saluggia (VC), Italy) The invention further relates to a methodof assay of an antibody-containing fluid of a person infected with HIVto determine the immunodeficiency of the person, where the sample issuspected to contain an antibody having a paratope specific to a TCR-Vβsubtype. A paratope is defined as that portion of an antibody whichenables the antibody to recognize and selectively bind to an epitope.The sample is prepared for contact with a binding agent specific to theparatope of the antibody. The prepared sample is contacted with thebinding agent under conditions suitable for binding of the antibody withthe binding agent. The extent of the binding is determined and acorrelation is made to the extent of the depletion. The extent of thebinding can be determined by conventional methods, as indicated above.

In another embodiment of the foregoing assay, the paratope of theantibody may be specific to an epitope which is present on a peptide andthe peptide is one of the following:Ser-Ala-Val-Ile-Ser-Gln-Lys-Pro-Ser-Arg-Asp-Ile-Cys-Gln-Arg-Gly-Thr-Ser-Leu-Thr;Gln-Leu-Gln-Glu-Thr-Glu-Asn-His-Lys-Lys-Arg-Phe-Ser-Ser-Gln-Cys-Pro;Gln-Asn-Leu-Ser-Ala-Ser-Arg-Pro-Gln-Asp-Arg-Gln-Phe-Ile-Leu-Ser-Ser-Lys;Asp-Gly-Tyr-Ser-Val-Ser-Arg-Ser-Lys-Thr-Glu-Asp-Phe-Leu-Leu;Pro-Arg-Asn-Arg-Ile-Thr-Lys-Ile-Gly-Lys-Arg-Ile-Met-Leu-Glu-Cys; orPro-Arg-His-Leu-Val-Arg-Arg-Arg-Gly-Gln-Glu-Ala-Arg-Leu-Arg-Cys.Peptides may be prepared by solid phase peptide synthesis techniques,recombinant DNA techniques or otherwise.

The paratope may also be specific to an epitope on a TCR-Vβ, which isone of the following: TCR-Vβ14, TCR-Vβ15, TCR-Vβ16, TCR-Vβ17, TCR-Vβ18,TCR-Vβ19, and TCR-Vβ20. The paratope of the antibody may also bespecific to an epitope on gp120 of HIV.

In another embodiment of the foregoing assay, the binding agent may bean antibody, an antibody fragment, a binding receptor, a bindingreceptor fragment, or a polypeptide containing fewer than 100 amino addswhere the polypeptide is capable of recognizing and selectively bindingto the paratope.

The invention further relates to a binding agent specific to a paratopeon a human antibody. The paratope on the antibody is specific to anepitope on a TCR-Vβ. The binding agent may itself be an antibody or anantibody fragment. The binding agent may be a binding receptor or abinding receptor fragment. The binding agent may also be described as apolypeptide containing fewer than 100 amino acids. The epitope on thebinding agent may be on an amino acid sequence which is one of thefollowing:Ser-Ala-Val-Ile-Ser-Gln-Lys-Pro-Ser-Arg-Asp-Ile-Cys-Gln-Arg-Gly-Thr-Ser-Leu-Thr;Gln-Leu-Gln-Glu-Thr-Glu-Asn-His-Lys-Lys-Arg-Phe-Ser-Ser-Gln-Cys-Pro;Gln-Asn-Leu-Ser-Ala-Ser-Arg-Pro-Gln-Asp-Arg-Gln-Phe-Ile-Leu-Ser-Ser-Lys;Asp-Gly-Tyr-Ser-Val-Ser-Arg-Ser-Lys-Thr-Glu-Asp-Phe-Leu-Leu;Pro-Arg-Asn-Arg-Ile-Thr-Lys-Ile-Gly-Lys-Arg-Ile-Met-Leu-Glu-Cys; orPro-Arg-His-Leu-Val-Arg-Arg-Arg-Gly-Gln-Glu-Ala-Arg-Leu-Arg-Cys.

The invention further relates to another method of assay of the fluidsof a person infected with HIV to determine the immunodeficiency of theperson. The method involves isolating a sample of a fluid from the HVinfected person where the sample is suspected to contain an antibodyhaving a paratope specific to an epitope on a TCR-Vβ and being bound tothe TCR-Vβ by the paratope to form an antibody-TCR-Vβ pair. The sampleis preferably blood or a blood fraction, although any bodily fluid whichmay contain an antibody-TCR-Vβ pair may be used. The sample is preparedfor contact with a binding agent which selectively binds to theantibody-TCR-Vβ pair. The binding agent does not bind to the antibodywhen the antibody is not bound to TCR-Vβ (ie. when the antibody is not acomponent of an antibody-TCR-Vβ pair) and the binding agent does notbind to the TCR-Vβ when the antibody is not bound to TCR-Vβ (ie. whenthe antibody is not a component of an antibody-TCR-Vβ pair). Theprepared sample is contacted with the binding agent under conditionssuitable for binding of the binding agent to any TCR-Vβ-pair. The extentof the binding is determined and a correlation is made to the extent ofthe depletion. The extent of binding is determined by conventionalmethods as described above.

In many of the foregoing assays a sample is prepared for binding with abinding agent. In the case where blood is used, it may be helpful toconvert the blood to serum. In some cases further processing of theserum (eg. by centrifugation to isolate certain cell types) may benecessary. Steps to correlate the extent of binding to the extent ofdepletion may be accomplished by running standards containing knownamount of analog, antibody or antibody-TCR-Vβ-pair in the assays anddeveloping a standard curve for use with experimental samples.

The invention further involves a method of treatment of a personinfected with HIV to attenuate or avert immunodeficiency in the personinfected with HIV. The method involves inoculating the person infectedwith HIV with a binding agent which is substantially homologous with anepitope on TCR-Vβ and which is reactive with a paratope in a freeantibody in the person's blood, where the paratope is specific to theepitope on the TCR-Vβ. Inoculation includes introduction of theimmunogen (ie. the binding agent) by ingestion (eg. orally) or byinjection by any conventional means (eg. intramuscular injection ortransfusion). The binding agent may be a polypeptide, a binding receptoror a binding receptor fragment as above. The binding agent may also bean antiidiotypic antibody or an antiidiotypic antibody fragment wherethe fragment possesses the ability to bind to a paratope in a freeantibody in the person's blood.

The invention further relates to another method of treatment of a personinfected with HIV to attenuate or avert immunodeficiency. The methodinvolves removing blood from the person infected with HIV, removingantibody from the blood of the person, the antibody having a paratopespecific to an epitope present on TCR-Vβ and reintroducing the bloodinto the person. The antibody can be removed by conventional means suchas pheresis.

The invention finally relates to a method of vaccination of a personinfected with HIV or a person at risk for infection with HIV in order toattenuate or avert immunodeficiency. The method involves inoculating theperson with an immunogenic substance capable of raising antiidiotypicantibodies in the person. Inoculation includes introduction of theimmunogen by ingestion (eg. orally) or by injection by any conventionalmeans (eg. intramuscular injection). The antiidiotypic antibodies arespecific to free antibodies containing a paratope specific to an epitopeon a TCR-Vβ.

Aspects of the invention may be illustrated by the following examples.

EXAMPLES Example 1 Lymphocyte Preparation from HIV+ Patients andControls

Ten ml of blood was drawn from six healthy volunteers (blood donors),from six symptomatic HIV+ patients with a history of major opportunisticinfection and CD4 lymphocyte counts of less that 200/mm³ (group 1, CDCstage IVC), from asymptomatic HIV+ patients with severe lymphocytesdepletion (CD4+ lymphocyte count <200/mm³), but without malignancy oropportunistic infection (group 2, stage CDC III), and from asymptomaticpatients who were HIV+ for more than 5 years and had normal lymphocytenumbers (group 3, stage CDC II). Table I shows the clinical parametersof these patients.

                  TABLE I    ______________________________________    Clinical Parameters of 14 HIV Seropositive Patients                                    CD4   Staging    Group   Patients  Age    Sex    n/mmc CDC    ______________________________________    1       A         22     F      25    IVC1            B         36     M      1     IVC1            C         28     M      17    IVC1            D         41     M      21    IVC1            E         46     M      13    IVC1            F         31     M      10    IVC1    2       G         30     M      100   III            H         30     M      196   III            I         26     M      147   III            J         30     M      156   III    3       K         37     M      851   II            L         25     F      852   II            M         27     M      827   II            N         38     M      825   II    ______________________________________     1 = AIDS patients     2 = Patients with severe immune depletion (CD4 <200/mm.sup.3), but withou     AIDS     3 = Patients with long history of HIV infection (>5 years) without a     severe depletion of CD4 lymphocytes

Peripheral blood mononuclear cells (PBMCs) were prepared from the abovepatient samples by Fycoll Hypaque gradient centrifugation at 2000 rpm,corresponding to 1645 g (Centrifuge Haereus, Omnifuge 2.0 RS, rotor2250).

Total RNA was prepared from about 0.5×10⁷ to 1×10⁷ PBMCs by theguanidine isothiocyanate-phenol-chloroform extraction method. PBMCs werewashed 3 times with Phosphate Buffered Saline (PBS) and resuspended in 1ml of a solution containing 4M guanidine isothiocyanate; 25 mM sodiumcitrate, pH 7.0; 0.5% sarcosyl; and 0.1M β-mercaptoethanol. 0.1 ml of 2Msodium acetate, pH 4.0; 1 ml of phenol and 0.2 ml of chloroform-isoamylalcohol mixture (1:49) were sequentially added to lysed cells and theresulting preparation was vigorously shaken, cooled on ice for 15 minand centrifuged at 10,000 g for 20 min at 4° C. The RNA, present in theaqueous phase, was mixed with 1 ml of isopropanol and left for 1 h at-20° C. The RNA pellet, resulted from a further centrifugation, wasdissolved in guanidine isothiocyanate solution and precipitated withisopropanol at -20° C. for 1 h. The RNA pellet was resuspended in 75%ethanol, vacuum dried and dissolved in 50 μl of H₂ O, 0.1%diethylpyrocarbonate.

Two μg of the RNA preparation was used to synthesize the first strand ofthe complementary DNA (cDNA) with a Riboclone cDNA Synthesis System,Promega Corp. (Madison, Wis.), using the instructions from themanufacturer.

For the analysis of the expression of human T cell receptor (TCR) Vα andVβ repertoire, 19 Vα, 22 Vβ, 2 Cα and 2 Cβ specific oligonucleotides,one of which was chosen to match a common sequence near to the 5' endsof the Cβ1 and Cβ2 genes, were prepared. The sequences of theoligonucleotides, reported in Table II, were obtained from previouslypublished reports and were selected to contain roughly the same G+Ccontent as the other primers.

                                      TABLE II    __________________________________________________________________________    T Cell Receptor β-Chain Primers (5' → 3'):    __________________________________________________________________________    V.sub.β 1,           GCACAACAGTTCCCTGACTTGCAC                               (SEQ ID NO:2)    V.sub.β 2,           TCATCAACCATGCAAGCCTGACCT                               (SEQ ID NO:3)    V.sub.β 3,           GTCTCTAGAGAGAAGAAGGAGCGC                               (SEQ ID NO:4)    V.sub.β 4,           ACATATGAGAGTGGATTTGTCATT                               (SEQ ID NO:5)    V.sub.β 5.1,           ATACTTCAGTGAGACACAGAGAAAC                               (SEQ ID NO:6)    V.sub.β 5.2,           TTCCCTAACTATAGCTCTGAGCTG                               (SEQ ID NO:7)    V.sub.β 6,           AGGCCTGAGGGATCCGTCTC                               (SEQ ID NO:8)    V.sub.β 7,           CCTGAATGCCCCAACAGCTCTC                               (SEQ ID NO:9)    V.sub.β 8,           ATTTACTTTAACAACAACGTTCCG                               (SEQ ID NO:10)    V.sub.β 9,           CCTAAATCTCCAGACAAAGCTCAC                               (SEQ ID NO:11)    V.sub.β 10,           CTCCAAAAACTCATCCTGTACCTT                               (SEQ ID NO:12)    V.sub.β 11,           TCAACAGTCTCCAGAATAAGGACG                               (SEQ ID NO:13)    V.sub.β 12,           AAAGGAGAAGTCTCAGAT  (SEQ ID NO:14)    V.sub.β 13.1,           CAAGGAGAAGTCCCCAAT  (SEQ ID NO:15)    V.sub.β 13.2,           GGTGAGGGTACAACTGCC  (SEQ ID NO:16)    V.sub.β 14,           GTCTCTCGAAAAGAGAAGAGGAAT                               (SEQ ID NO:17)    V.sub.β 15,           AGTGTCTCTCGACAGGCACAGGCT                               (SEQ ID NO:18)    V.sub.β 16,           AAAGAGTCTAAACAGGATGAGTCC                               (SEQ ID NO:19)    V.sub.β 17,           CAGATAGTAAATGACTTTCAG                               (SEQ ID NO:20)    V.sub.β 18,           GATGAGTCAGGAATGCCAAAGGAA                               (SEQ ID NO:21)    V.sub.β 19,           CAATGCCCCAAGAACTCACCCTGC                               (SEQ ID NO:22)    V.sub.β 20,           AGCTCTGAGGTGCCCCAGAATCTC                               (SEQ ID NO:23)    V.sub.β 21,           TTCTGCAGAGAGGCTCAAAGGACT                               (SEQ ID NO:24)    V.sub.β 22,           CTCAGTTGAAAGGCCTGATGGATC                               (SEQ ID NO:25)    V.sub.β 23,           CTCAGCTCAACAGTTCAGTGACTA                               (SEQ ID NO:26)    V.sub.β 24,           CCAATCCAGGAGGCCGAACACTTC                               (SEQ ID NO:27)    C.sub.β 3',           GTGCACCTCCTTCCCATT  (SEQ ID NO:1)    C.sub.β 5',           GTCCTGTGTTTGAGCCATCAGAA                               (SEQ ID NO:34)    C.sub.β NH.sub.2           ACCCAAAAGGCCACACTGGTGTGCCTGGCC                               (SEQ ID NO:35)    __________________________________________________________________________

All the primers were prepared using a DNA synthesizer (PCR Mate 391-EP;Applied Biosystem, Foster City, Calif.). Aliquots of cDNA (1 μl,corresponding to 1/25 of the initial amount of RNA) were analyzedseparately by amplification using TCR Vβ and a Cβ region specificprimers. One μl of each of the cDNAs was combined in 100 μl of reactionvolume with an amplification mixture containing 4 U of DNA polymerase(Tth DNA Polymerase from Thermus thermophilus HB8; Toyobo, Osaka,Japan); dNTP, at the final concentration of 250 μM each; 50 pmoles ofeach primer, in a buffer prepared with 67 mM Tris•HCl, pH 8.8; 16.6 mM(NH₄)₂ SO₄ ; 10 mM β-mercaptoethanol; 6.7 μM EDTA; 1.5 mM MgCl₂ and 170μg/ml of BSA.

The final reaction volume was brought to 100 μl with double distilled H₂O and the mixture was overlaid with 50 μl of mineral oil, heated inwater bath at 95° C. for 5 min and cooled rapidly on ice, in order todenature the DNA/RNA complexes. The reaction was taken through 40 cyclesin a thermal automatic cycler (Perkin-Elmer Thermal Cycler; Norwalk,Conn.), with each cycle consisting of 94° C. for 1 min (denaturationstep), 55° C. for 1 min (annealing step), 72° C. for 1.5 min (elongationstep).

Example 4 DNA Enzyme Immunoassay (DEIA)

The assay for the presence or absence of each amplification product wascarried out using an assay containing an antibody specific to doublestranded DNA (available as DEIA assay by Sorin Biomedica S.p.A.,Saluggia(VC), Italy). The expression of each V gene transcript wasoperationally defined by an optical density value obtained with the DEIAtest, performed using a Cβ or a Cα specific capture probe, mapping to aregion internal to the amplified cDNA. The test is based on a monoclonalantibody that recognizes double stranded, but not single stranded DNA.Thus, the antibody can only react with the solid phase if the amplifiedproduct is complementary to the immobilized probe. The sensitivity andthe specificity of the DEIA assay is similar to conventional Southernblot.

The CαNH₂ and CβNH₂ oligonucleotides used as capture probes (forsequences, see Table II) were modified and biotinylated at the 5' end.The modification was achieved by introducing a primary amino groupduring the synthesis using Aminolink 2 (Applied Biosystems). Five nmolof modified oligonucleotides, resuspended in 10 μl of double distilledH₂ O, were mixed with 17 μl of a solution of N-hydroxysuccinamidobiotinin N,N-dimethylformamide (145 mmol/l). After an overnight incubation,the biotinylated oligonucleotides were purified by filtration through aSephadex G-15 column (Pharmacia, Uppsala, Sweden) according to themanufacturer's instructions.

According to the DEIA assay protocol, streptavidin coated 96 microliterplates were incubated overnight at 4° C. with 5 ng/well of biotinylatedCαNH₂ or CβNH₂ oligonucleotides in 100 μl of TE (Tris 10 mM, pH 8.0; 1mM EDTA, pH 8.0). The solid phase was washed 5 times with 200 μl ofwashing solution (6.7 mM phosphate buffer, pH 6.1; 0.13M sodiumchloride; 0.004% Cialit and 0.1% Tween 20).

The amplification mixtures from Example 3 were denatured on a heat blockfor 10 min at 100° C. and then quickly cooled on ice. Twenty μl of theamplification product, diluted 5 times in hybridization solution (1×SSC, 2× Denhardt's solution, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA), wereadded to the coated wells and incubated for 1 h at 50° C. After 5 washeswith washing solution, each well received 100 μl of a 1/1000 dilution inPBS-10% Fetal Calf Serum (FCS) of a standard preparation of the 27 14-D9mAb, specific to double stranded DNA.

After 2 h incubation at 37° C. and 5 washes with washing solution, thebound antibody was revealed with 100 μl of HRP-labeled rabbit anti-mouseIgG antibody (ICN Biochemical, High Wycombe, Bucks, England) diluted1/20,000 in PBS-10% FCS. Following 1 h incubation at room temperatureand 5 washes, the wells received 100 μl of the chromogen/substratesolution (0.1M citrate buffer, pH 5, containing phenylenediaminehydrochloride and 10 μl of H₂ O₂) and the calorimetric reaction wasallowed to develop for 30 min at room temperature in the dark. Afterblocking with 200 μl of 1N sulfuric acid, the net absorbance was read ina spectrophotometer at 450 nm.

The expression of most of the known TCR Vα and Vβ genes was analyzed byamplification of mRNA from PBMCs obtained from 6 patients affected byAIDS and from 6 normal healthy individuals. Total RNA was separated fromeach sample immediately after collection and, at the time of analysis,was reverse transcribed in cDNA, as described in Example 1. Aliquots ofcDNA were amplified, as described in Example 3, with each of the 22 5'Vβ-specific sense primers and a 3' Cβ-specific antisense primer or witheach of the 19 5' Vα specific sense primers and a 3' Cα-specificantisense primer. Altogether, these primers are expected to cover mostof the sequenced human TCR Vα and Vβ genes. Although theoligonucleotides specific to Vβ21-24 were not used in the currentexample, one could also use these oligonucleotides as primers to expandthe scope of the analysis.

The comparative analysis of the Vβ repertoire in AIDS patients and innormal controls revealed the existence of important differences in thenumber of the Vβ genes expressed by the two groups FIG. 1. Expression ofthe TCR-Vβ genes in T cells from AIDS patients () and from normalcontrols(∘) is shown in the figure. The results are expressed asabsorbance at 450 nm from the DEIA test. The cut-off value was 0.20 andrepresents the mean value of 10 negative controls ±3 SD. The Vβ14, Vβ15,Vβ16 and Vβ18 sequences could not be detected in any of the cDNAsderived from lymphocytes of HIV+ patients. Similarly, Vβ17, Vβ19 andVβ20 were found to be expressed in only one of these cDNAs. All other Vβgenes analyzed were randomly represented in HIV+ patient cells.

The results reported in FIG. 2 demonstrate that there were no majordifferences between the Vα repertoires of normal individuals and of HIV+patients. Expression of the TCR-Vβ genes in T cells from AIDS patients() and from normal controls(∘) is shown in the figure, the results areexpressed as absorbance at 450 nm from the DEIA test. The cut-off valuewas 0.20 and represents the mean value of 10 negative controls ±3 SD.Most of the Vα genes were expressed in all samples and there was noevidence of important genomic or somatic deletions. Although the resultsare only qualitative, due to possible variations in the efficiency ofthe amplification reactions, it appeared that the relative abundance ofthe 19 Vα transcripts analyzed was highly variable in both group ofsamples. These variations are probably correlated to the positive andnegative selections processed that occur in the periphery of the immunesystem.

Example 5 Analysis of the Vβ repertoire by Southern Blot Hybridization

Vβ expression in T cells from individual AIDS patients (group 1 of TableI), and from one healthy control also of Table I, was also analyzed bySouthern blot hybridization with a ³² P labeled Cβ-specific probe. Afteramplification, the layer of mineral oil was removed and aliquots (10 μl)of each amplification reaction were analyzed by electrophoresis in 1%agarose gels. Bands were visualized by ethidium bromide staining andphotographed at 302 nm. The expected size of the amplification productsobtained was 129 base pairs (bp) for Vα-Cα products and 330 bp for Vβ-Cβproducts.

Electrophoresed amplified DNA was transferred to Hybond N nylon blottingmembrane (Amersham, Amersham, UK), following the manufacturer'sinstructions. Filters were incubated for 2 h at 65° C. inprehybridization solution containing 5× SSC (1× SSC: 0.15M NaCl, 0.015Msodium citrate), 5× Denhardt's solution (1× Denhardt's: 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% BSA), 0.1% SDS, 100 μg/ml salmon spermDNA and then hybridized overnight at 42° C. with ³² P-labeled Cβ5'specific probe at 1×10⁶ cpm/ml of hybridization solution. The membranewas then washed twice for 10 min at room temperature in 2× SSC, 0.1% SDSand used to expose X-OMAT, XAR X-ray film (Kodak).

The results of the experiment are shown in FIG. 3. FIG. 3A shows theresults of the DEIA assay of the amplified TCR-Vβ transcripts obtainedwith T cells from a normal control. The experiment was carried out asdescribed, using the CβNH₂ oligonucleotide shown in Table II as thecapture probe. FIG. 3B shows the results of Southern blot analysis ofamplified TCR-Vβ and Cβ transcripts obtained with T cells of a control(NC) and from AIDS patients (group 1, Table I) .

T cells from the control expressed all the Vβ genes, but there wereconsiderable differences in the level of the individual transcripts. Inthis sample Vβ15, Vβ16 and Vβ18 were the least expressed, while Vβ5.1and Vβ13.1 were the most abundant. The Vβ repertoire of the AIDSpatients was, in general, more restricted as compared to the normalcontrol, but the degree of this restriction varied considerably amongthe different samples. Patient B expressed only Vβ1, Vβ5.2 and Vβ6; allother transcripts were either absent or barely detectable. The next mostcompromised repertoire was the one of patient A, in which we detectedonly 8 of the 22 transcript tested. Patient D expressed the highestnumber of Vβ genes but, even in this case, the transcription of 8 Vβsegments could not be detected. The most striking result of thisanalysis was that, beside the individual variations, all samples lackedthe expression of a common set of Vβ genes comprising Vβ14, Vβ15, Vβ16,Vβ17, Vβ18, Vβ19 and Vβ20. The only exception was patient F thatexpressed Vβ19 and extremely low levels of Vβ17. These results establishthat HIV infection results in a severely compromised Vβ repertoire inwhich members of particular Vβ families are preferentially affected.

Example 6 Vβ mRNA Levels and Disease Progression

To study whether Vβ extinction correlates with the progression of HIVinfection to AIDS, we analyzed the expression of Vβ genes in two othergroups of HIV positive patients. Group A consisted of patients withsevere immune depletion (CD4<200 mmc), without the occurrence of theclinical manifestation of AIDs syndrome (CDC stage III). Group Bconsisted of patients with a long history of HIV infection (>5 years)that had retained a normal immunological profile (CDC stage II). Theresults reported in FIG. 4 show that the two groups display a distinctpattern of Vβ expression. The pattern of the group A (indicated by a"") patients strongly resembles the one seen in stage IVC1 AIDSpatients in which Vβ14, Vβ15, Vβ16, Vβ17, Vβ18, Vβ19 and Vβ20 are eithernot expressed or barely detectable. Conversely, the group B (indicatedby a "∘") displayed a pattern of Vβ expression more similar to the oneof normal controls, in which most of the Vβ genes were randomlyrepresented.

Example 7 Immunofluorescence Analysis of Cells

Fluorescein-conjugated mAb to CD3 and CD4 (T3 FITC and T4-FITC) werepurchased from Coulter Immunology, Hialeah, Fla., and thefluorescein-conjugated anti-TCR-specific antibodies (anti Vβ5, anti-Vβ6,anti-Vβ8 and anti-Vβ12) were purchased from T Cell Diagnostic, Inc.,Cambridge, Mass. Staphylococcal enterotoxins E, B and C2 (SEE, SEB andSEC2) were purchased from Serva Feinbiochemica, GmbH & Co. Heidelberg.

Peripheral blood mononuclear cells (PBMC) were prepared from heparinizedhuman peripheral blood samples from clinically healthy donors as inExample 1. Cells were placed at a concentration of 2×10⁶ /ml on petridishes coated either with 100 ng/ml of T3 mAb or with 10 ng/ml of SEE,50 ng/ml of SEC2 and 100 ng/ml of SEB. The enterotoxin SEB is known tostimulate T cells positive for Vβ3, Vβ12, Vβ14, Vβ15, Vβ17, and Vβ20;SEC2 is known to stimulate T cells positive for Vβ12, Vβ13.1, Vβ13.2,Vβ14, Vβ15, Vβ17, and Vβ20; and SEE is known to stimulate T cellspositive for Vβ5.1, Vβ6.1, Vβ6.2, Vβ6.3, Vβ8, and Vβ18. After 3 days ofculturing, the activated cells were washed and incubated overnight withor without two doses (67.2 and 6.72 ng/ml) of insolubilized gp120(Transgene, Stranbourg) in RPMI 1640 (GIBCO Laboratories, Grand Island,N.Y.). In another series of experiments 2×10⁶ lymphocytes werepreincubated overnight with 67.2 ng/ml of gp120 and then for 4 or 5 dayswith SEE (10 ng/ml), SEC2 (50 ng/ml) or SEB (100 ng/ml).

For staining, 5×10⁵ blasts (ie. PBMCs activated by enterotoxins SEB,SEC2, or SEE) in 100 μl of PBS, 2% FC3, were incubated for 30 min. at 1°C. with FITC-conjugated anti-CD3, anti-CD4, anti-CD8, and anti-TCR mAbs.All samples were analyzed on a EPICS C flow cytometer (Coulter) gated toexclude non viable cells. 2×10⁴ viable cells were accumulated forhistograms using logarithmic amplification of fluorescence intensity.Data are presented as percentage of positive cells after subtraction ofbackground staining with the fluorescent anti Ig reagent. Unstainedcells or cells stained with FITC-GαM Ig (Fab')₂ (Technogenetics,Trezzano sul Naviglio, Italy) were used as negative controls.

PBMCs from healthy individuals were first activated with thesuperantigens SEB, SEC2 and SEE that selectively expand those cellsbearing Vβ regions for which specific monoclonal antibodies areavailable. After 3 days of culturing the activated cells were washed andincubated overnight with or without various doses of insolubilizedgp120. Flow cytometric analysis with fluorescein-labeled antibodiesspecific to CD3, CD4, Vβ5, Vβ6, Vβ8 and Vβ12 revealed that preincubationwith gp120 did not change the membrane expression of CD3 and CD4 (FIG.5), but resulted in membrane down-modulation of some, but not all, Vβsegments and that the phenomenon was strictly dependent on theconcentration of gp120 added to the cultures (FIG. 6). Different Vβsegments were modulated in cell samples deriving from different donors,suggesting that polymorphic structures may be involved in thisinteraction.

In a second series of experiments PBMC were first incubated overnightwith or without gp120 and, after washing, recultured for five days inthe presence of SEB, SEC2, SEE. Flow cytometric analysis usingfluoresceined anti-Vβ antibodies revealed that, in some samples,preincubation with gp120 favored the expansion of those cells bearingVβ6 and Vβ8 but not of those bearing Vβ5 and Vβ12 (FIG. 7). Takentogether, these observations suggest that only some Vβ segments may actas a co-receptor for gp120.

Example 8 Autoimmunity and TCR-Vβ Components

A comparison of the amino acid sequences of gp120 and the known humanTCR segments carried out in our laboratories has revealed that gp120shares common motifs with some Vβ genes. Some of these motifs map in themost potentially antigenic region of the relevant Vβ segments. On thebasis of this evidence we constructed a first series of peptidescorresponding to the putative immunogenic regions of Vβ4(Ser-Ala-Val-Ile-Ser-Gln-Lys-Pro-Ser-Arg-Asp-Ile-Cys-Gln-Arg-Gly-Thr-Ser-Leu-Thr),Vβ19(Gln-Leu-Gln-Glu-Thr-Glu-Asn-His-Lys-Lys-Arg-Phe-Ser-Ser-Gln-Cys-Pro)and Vβ20(Gln-Asn-Leu-Ser-Ala-Ser-Arg-Pro-Gln-Asp-Arg-Gln-Phe-Ile-Leu-Ser-Ser-Lys).These peptides can be found in the region of greatest peptidehydrophilicity for each of the corresponding peptides (FIG. 8). Thechoice of these immunodominant epitopes of these TCR Vβ peptides wasdone with the aid of a computer program (PCGENE; Intelligenetics) usingthe Hopp and Wood algorithm. Alternatively, one could construct thefollowing series of peptides for use in the present invention: Vβ12(Asp-Gly-Tyr-Ser-Val-Ser-Arg-Ser-Lys-Thr-Glu-Asp-Phe-Leu-Leu), Vβ15(Pro-Arg-Asn-Arg-Ile-Thr-Lys-Ile-Gly-Lys-Arg-Ile-Met-Leu-Glu-Cys), andVβ18 (Pro-Arg-His-Leu-Val-Arg-Arg-Arg-Gly-Gln-Glu-Ala-Arg-Leu-Arg-Cys).

The peptides were synthetized by solid phase peptide synthesis nowdescribed. A polyamidic resin, functionalized with the C-terminal aminoacid (0.1 meq/g), was used as solid support. All of the amino acids werepentafluorophenyl esters preactivated and protected at the α-amino groupwith 9-fluorenilmethoxycarbonyl (Fmoc) and in the lateral chainsaccording to the nature of specific amino acid, 1-hydroxy-benzothriazole(HOBT) was used as catalyst and each coupling was conducted in dimethylformamide (DMF) with a 5 fold excess of amino acid for 2 hours. DMF wasused for wash and 20% piperidine/DMF for the deprotection of the α-aminogroups. At the end of synthesis the resin was washed successively withDMF, glacial acetic acid, tort-amylic alcohol, dietyl ether anddessicated. The peptide was cleaved from the resin with a mixture oftrifluoroacetic acid (TFA) and scavengers (anisole, phenol, ethandiol(EDT)) for 2 hours at room temperature (94% TFA, 5% anisole and 1% EDTfor Vβ4, Vβ15, Vβ18, and Vβ19; 95% TFA and 5% phenol for Vβ12 and Vβ20).The resin was separated from the solution by filtration, the filtratewas evaporated and the slurry obtained was precipitated with diethylether. The product was repeatedly washed with diethyl ether, thendissolved in glacial acetic acid and lyophilized. The peptides werepurified by RP-HPLC on a C18 column with a gradient of water andacetonitrile both containing 0.1% TFA. The composition of the peptideswas controlled by amino acid analysis with phenylisothiocyanate (PITC)pre-column derivatization.

The sera of AIDS patients (CDC stage: IV) and healthy HIV-individualswere then tested for immunoreactivity against this first series ofpeptides. Microtiter dishes were coated by treating with 200 μl of a 5μg/ml peptide solution in 0.05M carbonate buffer, pH 9.6 for one hour at37° C. The peptide solution was then removed and the wells overcoatedwith 300 μl of 0.2% BSA in 0.1M Tris•HCl buffer, pH 7.4 for two hours atroom temperature. The wells were then fixed with 300 μl of 10%saccharose, 4% PVP, 9% NaCl in water for one hour at room temperature.The fix solution was removed and the wells dessicated at roomtemperature overnight. The various sera were diluted 1/50 in PBS/10% FCSand 200 μl of the diluted sera were added to wells for one hour at 37°C. The sera were then removed and the wells washed with "wash buffer"from ETI-Kits (Sorin Biomedica; Saluggia, Italy). Polyclonal anti-humanIg-peroxidase conjugated was diluted 1/3000 in PBS/10% FCS and 200 μlaliquots were added to the wells for one hour at 37° C. The wells werethen washed three times with "wash buffer". Chromogen-substrate (100 μl,diluted 1:1 according to instructions) was added in the dark and allowedto incubate for 30 minutes at room temperature. The reaction was stoppedwith 0.1M sulphuric acid.

The results of the tests are shown in FIG. 9. Immunoreactivity of seraobtained from normal donors (right) and from HIV-infected patients(left) against the immonodominant peptides of 4, 19, and 20 is shown.Immunoreactivity against these peptides is found preferentially in thesera of AIDS patients. The specificity of these reactions was verifiedby inhibition experiments with the homologous peptides.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  57    - (2) INFORMATION FOR SEQ ID NO:  1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  1:    #  18              TT    - (2) INFORMATION FOR SEQ ID NO:  2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  2:    #                24ACTT GCAC    - (2) INFORMATION FOR SEQ ID NO:  3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi,Daniel - #e    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  3:    #                24CCTG ACCT    - (2) INFORMATION FOR SEQ ID NO:  4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  4:    #               24    - (2) INFORMATION FOR SEQ ID NO:  5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo; Primi,                   Daniele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  5:    #               24    - (2) INFORMATION FOR SEQ ID NO:  6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  25 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi,Daniel - #e    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  6:    #               25    - (2) INFORMATION FOR SEQ ID NO:  7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo; Primi,                   Daniele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  7:    #                24CTGA GCTG    - (2) INFORMATION FOR SEQ ID NO:  8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  8:    # 20               TCTC    - (2) INFORMATION FOR SEQ ID NO:  9:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  22 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  9:    #                 22CTC TC    - (2) INFORMATION FOR SEQ ID NO:  10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  10:    #                24ACGT TCCG    - (2) INFORMATION FOR SEQ ID NO:  11:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  11:    #                24AAGC TCAC    - (2) INFORMATION FOR SEQ ID NO:  12:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  12:    #                24TGTA CCTT    - (2) INFORMATION FOR SEQ ID NO:  13:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi, Da - #niele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb                   Sequences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  13:    #                24TAAG GACG    - (2) INFORMATION FOR SEQ ID NO:  14:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo;                   Primi,Daniel - #e    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  14:    #  18    - (2) INFORMATION FOR SEQ ID NO:  15:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                   Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  15:    #  18              AT    - (2) INFORMATION FOR SEQ ID NO:  16:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  16:    #  18              CC    - (2) INFORMATION FOR SEQ ID NO:  17:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  17:    #                24AGAG GAAT    - (2) INFORMATION FOR SEQ ID NO:  18:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell    Receptor                   Vb region - #)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  18:    #                24CACA GGCT    - (2) INFORMATION FOR SEQ ID NO:  19:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                (oligonucleotide usef - #ul in amplification of T Cell    Receptor                 Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                   Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  19:    #                24ATGA GTCC    - (2) INFORMATION FOR SEQ ID NO:  20:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  21 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                   Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  20:    #21                TTCA G    - (2) INFORMATION FOR SEQ ID NO:  21:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell                    Receptor - # Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng    #         oligonucleotide synthesis mac - #hine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  21:    #                24CAAA GGAA    - (2) INFORMATION FOR SEQ ID NO:  22:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                   (oligonucleo - #tide useful in amplification of T Cell    Receptor                   Vb region - #)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  22:    #                24CACC CTGC    - (2) INFORMATION FOR SEQ ID NO:  23:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #in amplification of T Cell Receptor                Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  23:    #                24AGAA TCTC    - (2) INFORMATION FOR SEQ ID NO:  24:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -     (vi) ORIGINAL SOURCE:  Synthesized using                   oligonucleot - #ide synthesis machine    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  24:    #                24AAAG GACT    - (2) INFORMATION FOR SEQ ID NO:  25:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -     (vi) ORIGINAL SOURCE:  Synthesized using                   oligonucleot - #ide synthesis machine    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  25:    #                24GATG GATC    - (2) INFORMATION FOR SEQ ID NO:  26:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -     (vi) ORIGINAL SOURCE:  Synthesized using                   oligonucleot - #ide synthesis machine    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  26:    #                24AGTG ACTA    - (2) INFORMATION FOR SEQ ID NO:  27:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                (oligonucleotide usef - #ul in amplification of T Cell    Receptor                 Vb region)    -    (iii) HYPOTHETICAL:  No    -     (vi) ORIGINAL SOURCE:  Synthesized using                   oligonucleot - #ide synthesis machine    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  27:    #                24AACA CTTC    - (2) INFORMATION FOR SEQ ID NO:  28:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:20 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:              (A) DESCRIPTION:  pepti - #de    -    (vii) IMMEDIATE SOURCE:  Chemical Synthesis    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  28:    - Ser Ala Val Ile Ser Gln Lys Pro Ser Arg    #                 10    - Asp Ile Cys Gln Arg Gly Thr Ser Leu Thr    #                20    - (2) INFORMATION FOR SEQ ID NO:  29:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:17 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:              (A) DESCRIPTION:peptide    -    (vii) IMMEDIATE SOURCE:Chemical Synthesis    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  29:    - Gln Leu Gln Glu Thr Glu Asn His Lys Lys    #                 10    - Arg Phe Ser Ser Gln Cys Pro                    15    - (2) INFORMATION FOR SEQ ID NO:  30:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:18 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:              (A) DESCRIPTION:peptide    -    (vii) IMMEDIATE SOURCE:Chemical Synthesis    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  30:    - Gln Asn Leu Ser Ala Ser Arg Pro Gln Asp    #                 10    - Arg Gln Phe Ile Leu Ser Ser Lys                    15    - (2) INFORMATION FOR SEQ ID NO:  31:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:15 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:              (A) DESCRIPTION:peptide    -    (vii) IMMEDIATE SOURCE:Chemical Synthesis    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  31:    - Asp Gly Tyr Ser Val Ser Arg Ser Lys Thr    #                 10    - Glu Asp Phe Leu Leu                    15    - (2) INFORMATION FOR SEQ ID NO:  32:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:16 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:              (A) DESCRIPTION:peptide    -    (vii) IMMEDIATE SOURCE:Chemical Synthesis    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  32:    - Pro Arg Asn Arg Ile Thr Lys Ile Gly Lys    #                 10    - Arg Ile Met Leu Glu Cys                    15    - (2) INFORMATION FOR SEQ ID NO:  33:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:16 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:              (A) DESCRIPTION:peptide    -    (vii) IMMEDIATE SOURCE:Chemical Synthesis    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  33:    - Pro Arg His Leu Val Arg Arg Arg Gly Gln    #                 10    - Glu Ala Arg Leu Arg Cys                    15    - (2) INFORMATION FOR SEQ ID NO:  34:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  23 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  34:    #                23ATCA GAA    - (2) INFORMATION FOR SEQ ID NO:  35:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  30 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Vb region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  35:    #           30     TGGT GTGCCTGGCC    - (2) INFORMATION FOR SEQ ID NO:  36:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  36:    #  18              CA    - (2) INFORMATION FOR SEQ ID NO:  37:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  37:    #                24TCTC TCAA    - (2) INFORMATION FOR SEQ ID NO:  38:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  38:    #                24GCAG TTAC    - (2) INFORMATION FOR SEQ ID NO:  39:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  21 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  39:    #21                TCCT A    - (2) INFORMATION FOR SEQ ID NO:  40:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  40:    #  18              GA    - (2) INFORMATION FOR SEQ ID NO:  41:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  41:    #                24ACAG AAGG    - (2) INFORMATION FOR SEQ ID NO:  42:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  21 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,    #Bettinardi, Alessandra; Puoti, Massimo; Primi,                   Daniele    #Depletion in HIV Infectiontive                   of T C - #ells That Bear Specific T Cell Receptor Vb Sequ    - #ences              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  42:    #21                ATAA A    - (2) INFORMATION FOR SEQ ID NO:  43:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    #         Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  43:    #                24CCTC AGAC    - (2) INFORMATION FOR SEQ ID NO:  44:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  44:    #                24CCGA GAAG    - (2) INFORMATION FOR SEQ ID NO:  45:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  45:    #                24AGAG CCCT    - (2) INFORMATION FOR SEQ ID NO:  46:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  21 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                (oligonucleotide usef - #ul in amplification of T Cell    Receptor                 Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  46:    #21                GTGT T    - (2) INFORMATION FOR SEQ ID NO:  47:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                (oligonucleotide usef - #ul in amplification of T Cell    Receptor                 Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  47:    #                24CGCA GACT    - (2) INFORMATION FOR SEQ ID NO:  48:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  48:    #                24ACAT TCCC    - (2) INFORMATION FOR SEQ ID NO:  49:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                (oligonucleotide usef - #ul in amplification of T Cell    Receptor                 Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  49:    # 20               CAAT    - (2) INFORMATION FOR SEQ ID NO:  50:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  50:    # 20               GGAA    - (2) INFORMATION FOR SEQ ID NO:  51:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  51:    #  18              AA    - (2) INFORMATION FOR SEQ ID NO:  52:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  52:    #                24GTCC TCAA    - (2) INFORMATION FOR SEQ ID NO:  53:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  18 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #in amplification of T Cell Receptor                Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  53:    #  18              GG    - (2) INFORMATION FOR SEQ ID NO:  54:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                (oligonucleotide usef - #ul in amplification of T Cell    Receptor                 Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  54:    #                24ACCC TTCC    - (2) INFORMATION FOR SEQ ID NO:  55:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  55:    #                24TGTC TGTG    - (2) INFORMATION FOR SEQ ID NO:  56:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                (oligonucleotide usef - #ul in amplification of T Cell    Receptor                 Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -      (x) PUBLICATION INFORMATION:    #Luisa; Sottini,THORS:  Imberti,                  Alessandra; B - #ettinardi, Alessandra; Puoti, Massimo;    Primi,                  Daniele    #Depletion in HIV Infectiontive                  of T Cells - # That Bear Specific T Cell Receptor Vb    Sequence - #s              (C) JOURNAL:  Science              (D) VOLUME:  254              (E) ISSUE:  5033              (F) PAGES:  860-862    #November 8, 1991LICATION DATE:    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  56:    #                24CCCT GCCG    - (2) INFORMATION FOR SEQ ID NO:  57:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  30 base - #s              (B) TYPE:  Nucleic A - #cid              (C) STRANDEDNESS:  Sing - #le              (D) TOPOLOGY:  Linear    -     (ii) MOLECULE TYPE:  Other nucleic aci - #d    #useful in amplification of T Cell Receptor                  Va region)    -    (iii) HYPOTHETICAL:  No    -      (v) ORIGINAL SOURCE:  Synthesized usi - #ng                   oligonucleot - #ide synthesis machine    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  57:    #           30     TGCC TATTCACCGA    __________________________________________________________________________

I claim:
 1. A method of binding free antibodies in serum of a personinfected with HIV comprising:(a) providing a person infected with HIV,the person having serum suspected of containing free antibodies, thefree antibodies having a paratope capable of binding to an epitope on aTCR-Vβ; (b) providing a binding agent which is substantially homologouswith the epitope on the TCR-Vβ, the binding reagent thus being reactivewith the paratope on the free antibodies; and (c) administering thebinding agent to the person.
 2. The method of claim 1 wherein thebinding agent is selected from the group consisting of a polypeptide, abinding receptor and a binding receptor fragment.
 3. The method of claim2 wherein the binding agent is an antiidiotypic antibody orantiidiotypic antibody fragment.
 4. The method of claim 2 wherein thebinding agent is a polypeptide selected from the group consisting of:(a)Ser-Ala-Val-Ile-Ser-Gln-Lys-Pro-Ser-Arg-Asp-Ile-Cys-Gln-Arg-Gly-Thr-Ser-Leu-Thr(SEQ ID NO: 28); (b)Gln-Leu-Gln-Glu-Thr-Glu-Asn-His-Lys-Lys-Arg-Phe-Ser-Ser-Gln-Cys-Pro (SEQID NO: 29); (c)Gln-Asn-Leu-Ser-Ala-Ser-Arg-Pro-Gln-Asp-Arg-Gln-Phe-Ile-Leu-Ser-Ser-Lys(SEQ ID NO: 30); (d)Asp-Gly-Tyr-Ser-Val-Ser-Arg-Ser-Lys-Thr-Glu-Asp-Phe-Leu-Leu (SEQ ID NO:31); (e) Pro-Arg-Asn-Arg-Ile-Thr-Lys-Ile-Gly-Lys-Arg-Ile-Met-Leu-Glu-Cys(SEQ ID NO: 32); and (f)Pro-Arg-His-Leu-Val-Arg-Arg-Arg-Gly-Gln-Glu-Ala-Arg-Leu-Arg-Cys (SEQ IDNO: 33).
 5. The method of claim 1 wherein said TCR-Vβ is selected fromthe group consisting of TCR-Vβ14, TCR-Vβ15, TCR-Vβ16, TCR-Vβ17,TCR-Vβ18, TCR-Vβ19 and TCR-Vβ20.
 6. The method of claim 1 wherein saidepitope is on an amino acid sequence selected from the group consistingof:(a)Ser-Ala-Val-Ile-Ser-Gln-Lys-Pro-Ser-Arg-Asp-Ile-Cys-Gln-Arg-Gly-Thr-Ser-Leu-Thr(SEQ ID NO: 28); (b)Gln-Leu-Gln-Glu-Thr-Glu-Asn-His-Lys-Lys-Arg-Phe-Ser-Ser-Gln-Cys-Pro (SEQID NO: 29); (c)Gln-Asn-Leu-Ser-Ala-Ser-Arg-Pro-Gln-Asp-Arg-Gln-Phe-Ile-Leu-Ser-Ser-Lys(SEQ ID NO: 30); (d)Asp-Gly-Tyr-Ser-Val-Ser-Arg-Ser-Lys-Thr-Glu-Asp-Phe-Leu-Leu (SEQ ID NO:31); (e) Pro-Arg-Asn-Arg-Ile-Thr-Lys-Ile-Gly-Lys-Arg-Ile-Met-Leu-Glu-Cys(SEQ ID NO: 32); and (f)Pro-Arg-His-Leu-Val-Arg-Arg-Arg-Gly-Gln-Glu-Ala-Arg-Leu-Arg-Cys (SEQ IDNO: 33).